The techniques of genetic engineering have been successfully applied to the pharmaceutical industry, resulting in a number of novel products. Increasingly, it has become apparent that the same technologies can be applied on a large scale to the production of enzymes of value to other industries. The benefits of achieving commercially useful processe's through genetic engineering are expected to include cost savings in enzyme production, productions of enzymes in organisms generally recognized as safe which are suitable for food products, and specific genetic modifications at the genomic level to improve enzyme properties, such as thermal stability and performance characteristics, as well as those which would increase the ease with which the enzyme can be purified.
Glucose oxidase is the enzyme which catalyzes the oxidation of glucose to gluconic acid with the concomitant production of hydrogen peroxide. The enzyme has many industrial uses, including its use in desugaring eggs, in the removal of oxygen from beverages, moist food products, flavors, and hermetically sealed food packages, and in the detection and estimation of glucose in industrial solutions, and in body fluids such as blood and urine.
Glucose oxidase was first isolated from cells of Aspergillus niger by Muller [Biocehmische Zeitschrift (1928), 199, 136-170 and (1931), 232, 423-424], and was also extracted from A. niger by Franke and Deffner [Annalen der Chemie (1939), 541, 117-150]. The production of glucose oxidase from cells of species of Penicillium chrysogenum, Penicillium glaucum, Pencillium purpurogenum, Aspergillus niger and Aspergillus fumaricus, has been described by Baker, in U.S. Pat. No. 2,482,724. A method for preparing glucose oxidase in which glucose oxidase-producing strains of the genera Aspergillus and Penicillium are cultivated in medium having a low carbohydrate content is described in U.S. Pat. No. 3,701,715. The enzyme from Aspergillus niger (A. niger) has been purified to a high degree of purity, and reportedly has a molecular weight of approximately 150,000, an isoelectric point of 4.2, and a flavin adenine dinucleotide (FAD) content of 2 FAD per mole. Pazur and Kleppe (1964), Biochemistry 3, 578-583. The amino acid composition of the enzyme from A. niger, as, well as its identity as a glycoprotein are also known. Pazur et al. (1965), Arch. Biochem. Biophys. 111, 351-357. However, neither the amino acid sequence of glucose oxidase, nor the nucleotide sequence encoding it are known.
A problem with utilizing glucose oxidase isolated from its native source is that the organisms which produce the enzyme may have contaminants which are deleterious for certain uses of the desired protein. For example, glucose oxidase is used for the commercial preparation of foodstuffs. However, A. niger, which is a major source of commercially prepared enzyme, is highly allergenic, and is not approved for use in food. Moreover, stringent purification procedures may be relatively expensive since glucose oxidase is primarily an intracellular enzyme. These problems could be solved by producing glucose oxidase in recombinant systems.
Fungal enzymes have been expressed from recombinant vectors. Glucoamylase from Aspergillus [Innis et al. (1985), Science 228, 21-26] and endoglucanase I from Trichoderma reesei [Van Arsdell et al. (1987), Biotechnology 5, 60-64] have been expressed in Saccharomyces cerevisiae.
References Cited in Following Text
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Disclosure of the Invention
The present invention provides a cDNA sequence encoding glucose oxidase (GO) from a fungal source of the genus Aspergillus, and more particularly from A. niger. Knowledge of this sequence allows the expression in recombinant systems of polypeptides substantially similar to GO, including GO, analogs of GO, and fragments of GO. Surprisingly, relatively large amounts of the enzyme are produced in and secreted from yeast cells, when the cells are transformed with an expression vector encoding GO, and grown under conditions allowing expression of the enzyme. The secretion may be under the control of either yeast secretory sequences, or the prepro sequence of GO encoded in A. niger.
The cDNA sequence provided herein also allows for the isolation of GO-encoding sequences from other sources, which can also be used for the production of recombinant GO. These other sources may be of any origin wherein the enzyme is naturally encoded, but will be particularly fungal sources, wherein the GO-encoding sequence contains at least 8 base pairs, preferably 20 base pairs, and even more preferably at least 40 base pairs which are highly homologous (i.e., have at most a one base mismatch in complementary sequences) to a comparable sequence in FIG. 5B. Alternatively, the GO isolated from the source other than A. niger may have a sequence of at least about 4 amino acids, homologous to that of the A. niger GO sequence encoded in the cDNA sequence in FIG. 5B.
The polypeptides expressed in yeast transformed with expression vectors encoding the GO cDNA have been examined, and the surprising result obtained that the products were hyperglycosylated, and that the hyperglycosylation of the recombinantly produced polypeptide has little or no effect on enzymatic activity, as compared to native GO, but that the recombinant product exhibited increased thermostability.
Another surprising result is that removal of the carbohydrate residues from both recombinantly produced GO and native GO apparently does not inhibit enzymatic activity.
Still another surprising result is that although native GO is present in A. niger in relatively large amounts, the mRNA encoding it is relatively rare in A. niger cells during log-phase growth.
Yet another surprising result is that an analog of GO, i.e., a mutein, exhibits increases thermostability relative to the native molecule from A. niger and to its recombinant counterpart expressed in yeast.
Accordingly, one aspect of the invention is a recombinant vector comprising a polynucleotide sequence encoding a polypeptide substantially similar to glucose oxidase (GO), essentially free of other vectors that do not encode GO.
Another aspect of the invention is a host cell transformed with a recombinant polynucleotide comprising a sequence encoding a polypeptide substantially similar to GO.
Yet another aspect of the invention is non-native polypeptide substantially similar to GO.
The invention includes a method of producing a recombinant polypeptide substantially similar to GO, comprising:
(a) providing a population of transformed cells containing a recombinant vector which is comprised of a coding sequence for a polypeptide substantially similar to GO operationally linked to sequences allowing expression of said coding sequence in said cells;
(b) growing said population of transformed cells under conditions whereby said polypeptide substantially similar to GO is expressed; and
(c) recovering said polypeptide substantially similar to GO.